Category Archives: Biology

One of the things we have been asked several times at the Royal Society Summer Science Exhibit is whether butterflies always have symmetrical wing patterns. This is almost always the case, because the wing patterns are hard-wired in the genome, and all cells on both wings have the same genetic code. However there are rare exceptions, where a single egg gets fertilised by two sperm, or a single individual developed from two genetically distinct cell lineages. These rare specimens are known as gynandromorphs, and can tell us something about wing development. Here, Martin Thompson describes a few specimens of gynandromorphs in the genus Papilio.

Mosaic gynandromorph of Papilio dardanus

This is a sex mosaic (‘mosaic gynandromorph’) Papilio dardanus. The left wing is mostly the pattern of the female form cenea, but the right wing shows lots of yellow patches of male pigment. There is even a segment at the bottom corner with male patterning and even a little bit of a tail.

P. dardanus female form cenea

P. dardanus male

P. dardanus female form hippocoon

It is thought that this kind of pattern arises from the loss of one of the sex chromosomes from one of the two daughters of a cell division, or possibly when an the egg contains an incorrect number of sex chromosomes.

In butterflies and moths, the sex determination system is different to humans – males have two identical sex chromosomes (ZZ rather than XY) and it is the females with two different sex chromosomes (ZW contrasting with XX in humans). If this loss happens very early in development whilst the embryo is only a few cells, the result can be a striking bilateral gynandromorphy, with one half of the body male and one half female:

Bilateral gynandromorph – right hand side mostly female pattern, left hand side male

However, if the error occurs later in development, the result will be a mosaic as in the first picture. Isn’t biology cool?!
Mosaics such as these can teach us about how wings are formed and patterned when the butterfly is still a pupae. By looking at a large number of mosaic butterflies, scientists have found that there are several ‘compartments’ to the wing: that there are boundaries which cells never cross during development. These boundaries are usually invisible, but in mosaic gynandromorphy wings, each differently-coloured patch arises as a handful of cells. When we see lots of mosaic patches with similar, sharply-defined and straight boundaries, this tells us about the position of the wing compartments.

Notice that the region where black meets yellow on the forewings is a straight and clear line. This marks a compartment boundary within the wing.

Every living animal and plant starts its life as a single cell. The cell then divides many times, and the end result is a fully functioning organism, like a rose bush or a horsefly or you or Brian Blessed. A human is made of quadrillions of cells, but they all come from that one initial fertilized egg. All (or at least most) of the information that makes you you can be compacted town to one nucleus of one single cell, and then read out in such a way that when that one cell starts to divide quadrillions of times, the new cells congeal into an all-signing, all-dancing organism-y thing. I think this is pretty awesome, and thinking about these problems has led me to working on a PhD studying Heliconius butterflies. This post is a personal perspective on this problem and how I came to study butterflies.Joe Hanly….or Henry Walter Bates?

The processes that make an animal from a single egg are controlled in such a way that they will always make a pretty accurate version of that animal. This process is really repeatable. Hold your hands together. They’re pretty much exactly the same size and shape, correct? In fact, I’d hazard a guess that your hands are more or less the same shape as the hands of all the other humans you know, too. All of the hands of all humans, including your own two hands, were put together completely independently of each other, yet in most cases they are pretty much perfectly formed (apologies to readers with polysyndactly. And to amputees).

How does that work? How does a hand come to be a hand, and what are the physical mechanisms that can build hands so perfectly and repeatably? How about feet, how do they happen – I guess in a similar way to hands, but with some differences? How does one make a face? I be that’s probably pretty different from the way you make a hand, right? Lungs? Wings? Shells? Roots? I’ve always wondered about how all this stuff gets put together from one egg. I always enjoyed learning about science at school, and studied Biology, Chemistry, Maths and History for my A-levels, and so when I was 17 and I actually had to start deciding what I wanted to do with my life, I decided to go and do a course in Developmental Biology at the University of Manchester.

While I was there, I actually learnt about how hands and faces and lungs and other bits develop, and why we think the process is so symmetrically perfect and repeatable. I’m not going to talk about that here (if you’re interested in these things, then I’ve put some useful links below), but while I was learning about this, I started realise there were even more questions I hadn’t really even considered before.

Firstly, I got really interested in gene regulation. For living things to function, they need to make proteins from genes. While some of these proteins need to be present in all places at all times (for example, the genes important for respiration) others need to be turned on or off in specific places and at specific times, for example, the proteins that make melanin pigments, or the proteins that form the structure of bone. If the proteins that make bones were present everywhere throughout your body, you’d be in trouble. It turns out that while we know quite a bit about the process of turning genes on and off, there is still a lot to be found.

I also got really interested in the way that gene regulation can relate to evolution. Over time, the physical form that a species develops will change in response to selection. Sometimes, these changes might involve the evolution of entirely new proteins or changes to existing proteins, but it turns out that the simplest and most common change that leads to differences in morphology actually affect the ways that genes are turned on and off.

When I finished my degree I decided that I wanted to continue studying science, so I applied for funding for a PhD from the Wellcome Trust, and came to study at the University of Cambridge. I’m working on Heliconius because it turns out to be a good system for investigating how differences in gene regulation can evolve, as we have one species with many different pattern forms, and we can use these patterns to gain an understanding of how the genetics which control phenotypic differences work. I’ll talk more about this in another post, though.

This year the annual Heliconius 2014 meeting will be hosted by the University of Puerto Rico, Rio Piedras campus from June 4th to June 6th. The meeting will be immediately prior to the SMBE meeting in San Juan, Puerto Rico (http://www.smbe.org/).

You can find a brief description of the Heliconius 2014 meeting at the following webpage: http://heliconius.wix.com/heliconiusmeeting

Please register using the form found on the website (http://heliconius.wix.com/heliconiusmeeting).

Breakfast and lunch will be provided for the three days of the meeting and special rate at the UPR housing ($50 per night) will be offered by request (see form on the webpage) to the participants. Possibility to extend the stay at the University housing will also be offered to the participants if requested (especially for the ones that will present at the SMBE meeting).

This was one of the first papers to show that evolution can happen at different rates in different parts of the genome. It seemed strange to us, spoiled as we are by whole genome sequences, that this was considered a significant finding as late as 1979, but it’s difficult to reconstruct the context. We’d be interested to hear from anyone who was around at the time.

Mayr, for example, had argued for ‘genetic revolutions’ following the isolation of a founder population:

Isolating a few individuals (the “founders”) from a variable population which is situated in the midst of the stream of genes which flows ceaselessly through every widespread species will produce a sudden change of the genetic environment of most loci. This change, in fact, is the most drastic genetic change (except for polyploidy and hybridization) which may occur in a natural population, since it may affect all loci at once. Indeed, it may have the character of a veritable “genetic revolution”. Furthermore, this “genetic revolution”, released by the isolation of the founder population, may well have the character of a chain reaction. Changes in any locus will in turn affect the selective values at many other loci, until finally the system has reached a new state of equilibrium.

Heliconius species are usually more like the variable populations Mayr describes than the founder populations. However, single species can have many different wing patterns. By 1979, it was well known that these wing patterns were controlled by a handful of genetic loci, through a long series of genetic crosses showing the perfect segregation of loci with different wing pattern elements: red rays, yellow bands and so on (summarised in Sheppard 1985). Species such as Heliconius erato have races spread across South America with very different patterns, and yet these races can interbreed.

How much of the genome was responsible for these wing pattern differences? The method of choice for answering this question in the 1970s was allozyme electrophoresis, as DNA sequencing was still in its infancy. Proteins with variations in amino acid composition carry different electrical charges. Varying proteins could therefore be separated by running them on a gel. By 1979, a large library of protein variations had been reported in a wide variety of species (documented in detail in Chapter 3 of Lewontin’s The Genetic Basis of Evolutionary Change, available as full free PDFs).

Turner et al were the first authors to test allozyme variation in Heliconius. They took a set of 17 enzymes and tested them for variations in eight species of Heliconius, including multiple races of many species. They showed that none of the enzymes segregated with colour pattern.

For example, wing patterns in Heliconius erato were known to be controlled by between one and seven genetic loci, but the sampled populations of erato were found to be between 93 and 99% identical based on the selected allozymes. This was very similar to the genetic identity seen in Heliconius sara, a species with very few races and only minor wing pattern differences.

The enzymes chosen were the ones that were available, with no intentional bias towards genes that may or may not have been involved in wing patterning. From our point of view, it was unsurprising that such enzymes would not necessarily be involved in wing patterning. But perhaps this was surprising in 1979; perhaps it was expected that loci unrelated to colour patterning would be ‘carried along’ with sharp variation in leading colour pattern loci, following lines of thought like Mayr’s. After all, it is not so different from the ‘islands of speciation’ arguments we are having today.

It may be that we are now used to the idea that only small regions of a few chromosomes are responsible for the variation in colour patterns, with the rest of the genome being mostly very similar and freely flowing between Heliconius races. But we are definitely still struggling with the idea that different parts of the genome can evolve at different rates, under different contraints. And with that, I have to get back to thinking about ABBA-BABA windows…

After we got the Heliconius melpomene genome, I thought it’d be easy to find out about one’s favourite gene on our genome browser or on ENSEMBL. Unfortunately, orthologous gene names for Heliconius don’t seem to have made it through to the public databases.

However, I recently went to a talk by Rob Waterhouse, who runs a website called OrthDB which classifies genes into orthologous groups. http://www.orthodb.org/ This provides one method for finding a Heliconius gene by name. OrthoDB obtained the Heliconius melpomene data from ENSEMBL, and then classified them into their orthologous groups, so you can search for gene names really easily there, and easily find genes, gene names, and their synonyms used in related species.

In the course of his work incorporating the Heliconius genome into OrthoDB, Rob Waterhouse also ran an analysis to find orthologous gene groups that really should be in the Heliconius genome (on the basis that they’re in most other related species), but that were not found by the method Jamie Walters used to annotate the genome, Maker. He forwarded this list to Jamie, but I don’t think any use was made of it. As he’s shared it with me too, I thought I’d make it public for those who are interested. I’ve attached it here.

H. melpomene laying an egg on its Passiflora menispermifolia hostplant

Unlike their male counterparts, female Heliconius butterflies have taste receptors on their legs in order to pick the best plants on which to lay their eggs.

Female Heliconius butterflies have taste receptors next to spikes on their legs in order to spear and ‘taste’ plants to find the most beneficial ones on which to lay their eggs, new research reveals. As male Heliconius butterflies do not lay eggs, they have no taste receptors on their legs. The research was published today, 11 July, in the journal PLoS Genetics.

For the research, the scientists studied the genes that code for the taste receptor proteins. Using new high-throughput sequencing methods, they were able to identify genes expressed at very low levels, including the great diversity of taste receptor genes unique to female Heliconius butterflies.

Because, unlike their parents, caterpillars cannot fly away to find a more suitable plant, it is imperative that the female butterflies choose the best host plant for their eggs or risk the survival of their offspring. The proteins that are coded for by the taste receptor genes enable the female butterflies to identify the most advantageous plants on which to lay their eggs.

Dr Chris Jiggins, lead author of the paper from the University of Cambridge said: “It appears that a new set of taste receptor genes have evolved to help identify toxic plants and are used by females to find the plant that will increase their caterpillars chance of survival.”

It is a long-standing hypothesis that butterflies are so diverse partly because of the complicated evolutionary arms race with the plants that their larvae eat – as plants develop new ways to prevent being eaten, butterflies develop new ways to eat plants.
For example, Heliconius butterflies evolved in a way that allows them to feed on the highly-toxic, cyanide-containing leaves of passion flower vines.

The Heliconius butterflies have not only evolved to overcome the plant’s defences, but can now even synthesise their own cyanide-containing compounds that protect them from predators.

Thanks to everyone who came to the Consortium meeting in Boston. It was a great success with lots of exciting biology and some rather arcane discussions of databasing problems. Thanks in particular to Bill Gelhart and others from FlyBase who came along to give advice, and Jim Mallet and Nikki Hughes for organising and hosting us. Here is the group photo and a list of everyone who attended.

It is an exciting time to be working on Heliconius butterflies, as new sequencing technologies allow us to get closer and closer to the genes underlying colour pattern variation. We’re going to start featuring new Heliconius papers on the blog, to highlight new findings and put them into context. In this post, we’re going to look at a new paper in Genome Research that describes the region of the genome that produces red patterns in Heliconius erato and reveals how this region varies between H. erato and its mimic, Heliconius melpomene [1].

Red bands and rays are one of the key features of Heliconius wing patterns and occur repeatedly in mimics across South America. For example, Heliconius melpomene and Heliconius erato mimic each other in many different countries, often sharing either bands or rays (see picture above [2]). For over a century, researchers have been trying to understand how this mimicry could evolve. One of the major parts of this puzzle is to discover the genes that produce the red patterns and how they vary between species.

It has been known for many years that these features are controlled by one large region of the genome less than one megabase long, known as the B locus in H. melpomene and the D locus in H. erato [3]. In recent years, we have been making great progress in describing and understanding this region. In 2010, the whole region was sequenced using bacterial artificial chromosomes (BACs) in H. melpomene and H. erato [4,5]; the region is around 700 kilobases long in H. melpomene and 1 megabase long in H. erato, and contains around 20 genes, which are not only the same genes in both species but are also conserved in the same order.

We now know that the optix gene, which is found within the B/D region, is expressed on the wing wherever red pattern elements are expressed across many different Heliconius species, and that genetic variations in optix are strongly associated with variations in red patterns in multiple species [6]. We also know that this gene has evolved to produce red patterns separately in H. melpomene and H. erato, converging on the same phenotype but differing genetically [2].

We now really want to know what’s happening across the whole B/D region. Are there any other genes strongly associated with red band patterns? What genetic variations in H. melpomene and H. erato exist in the wild? To answer these questions, we need to sequence the whole B/D region in many butterflies from each species. Unfortunately, until recently it has not been possible to do this. BAC sequencing of one copy of the whole B/D region was very laborious and expensive; it would be very difficult to sequence tens of butterflies this way. A short 800 bp fragment of optix was sequenced in many butterflies using polymerase chain reactions (PCRs) to associate optix with red patterns, but over 1000 similar experiments would be required to cover the whole region.

With next generation sequencing, we can now sequence whole genomes of many butterflies and use these sequences to study particular regions of the genome like B/D. The new paper in Genome Research by Megan Supple and colleagues reports on the sequencing of short reads from the whole genomes of 45 H. erato butterflies from four hybrid zones across South America using next generation sequencing. By taking many butterflies with rayed patterns and red banded (‘postman’) patterns, and looking for variations in the D locus sequences, we can see what’s going on across the whole region.

Next generation sequencing data is still fairly new, and as with many scientific projects the scientists usually learn how to analyse their data during the project. As Megan says, “I was just handed a large pile of data. At that point, I had no idea how to go about analyzing next-gen sequences. It was quite a learning process.”

It is not yet possible to sequence whole chromosomes in one go; current technologies can only produce short reads a few hundred bases long. The raw sequence data for the 45 H. erato genomes is hundreds of millions of 100 base pair (bp) sequence reads. The H. melpomene genome has been assembled, but the H. erato genome has not, so the first step of the analysis was to align the 100bp reads to the existing BAC sequence of the erato D region. By comparing reads between different butterflies at the same locations, variations in the D locus could be identified. This is done with tools like the Genome Analysis Tool Kit, a suite of software written to analyse human genome data sets such as the 1000 Genomes Project data, but as DNA is the same in butterflies and humans, we can use the same software to study the evolution of mimicry.

Megan found the same strong association with optix shown in previous papers, but also found a very strong association in a 65 kb long region upstream of optix, which does not appear to contain any genes. This strongly suggests that there are sequences that regulate other genes in this region, which are responsible for turning the genes with red patterns on and off. In the H. melpomene genome paper, this same region was shown to vary considerably between H. melpomene subspecies [7].

“What surprised me the most was that analyzing H. erato and H. melpomene separately identified almost the same exact region. I did not expect that the boundaries identified in H. melpomene would almost perfectly coincide with the boundaries in H. erato. That indicates that we have hit the boundary of something important – we believe that those boundaries are close to important functional regions that are under strong divergent selection.”

So are these regions actually the same in both species? Apparently not. In H. erato, there are 76 single base variations with one variant perfectly associated with the postman pattern and the other associated with rays; in H. melpomene, there are 430 such variations. When the sequences of the whole regions are compared, the sequences from H. melpomene individuals with rayed patterns do not group together with sequences from H. erato rayed individuals, and H. melpomene postman individuals do not group with H. erato postman individuals.

This strongly indicates that, although the H. melpomene and H. erato butterflies look very similar, the genetic sequences producing the patterns are not the same, and H. melpomene has evolved the pattern independently from H. erato. Megan Supple: “It was really compelling to see how perfectly the sequences in this region cluster by pattern within each species, but always keep the two mimics separate.”

Now the architecture of the B/D region is clear, and the full sequences and variations are available, we can search for the functional variants that produce the different red patterns. This will take some time, as there are 65,000 bases to search through, and confirming the function of any candidate sequences will require some difficult experimental work. But this new paper brings us very close to finally identifying these variants, and to providing a mechanical explanation for the evolution of red pattern mimicry in Heliconius.